Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.
Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.
Park
K
K
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.
Debyser
Z
Z
Tabor
S
S
Richardson
C C
CC
Griffith
J D
JD
D017104:Bacteriophage M13 / Q000235:genetics*; D017123:Bacteriophage T7; D001483:Base Sequence; D019921:Biotinylation; D004247:DNA; D004265:DNA Helicases / Q000378:metabolism / Q000648:ultrastructure; D019915:DNA Primase / Q000378:metabolism / Q000648:ultrastructure; D017931:DNA Primers; D004261:DNA Replication; D004270:DNA, Circular / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000648:ultrastructure; D004279:DNA, Viral / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism / Q000648:ultrastructure; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism / Q000648:ultrastructure
D017931:DNA Primers; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003845:Deoxycytosine Nucleotides; C022262:Okazaki fragments; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D019915:DNA Primase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases